Title: Improved Dosing and Refinement of a Cardiac Targeted Gene Therapy in a Swine Heart Failure Model Abstract: The goal of this Direct to Phase II SBIR is to build on prior progress developing BB-R12 gene therapy for heart failure to 1) increase the scale of vector production and using GMP to support IND-enabling studies and clinical trials, 2) expand beyond the POC data, performing a 2nd pig infarct/heart failure study with more statistical power and a longer period infarct and treatment, 3) improve the delivery and distribution in the heart, and 4) generate additional efficacy and safety data needed for progression to clinical studies and demonstrating that a novel, myofilament targeted gene therapy can reduce, halt, or reverse the decline in cardiac function following myocardial infarction (MI) and heart failure. ~1 million Americans suffer MI yearly, accounting for most of the >600,000 annual new cases of diagnosed heart failure (HF). With the exception of transplantation, current treatments are merely palliative and fail to restore cardiac function. Thus, treatments that halt progression to failure or improve function are needed and could significantly lower healthcare costs, especially with an aging US population. Dr. Michael Regnier, a BEATBio Co-Founder and Co-Investigator, has demonstrated that small increases in cardiomyocyte 2-deoxy ATP (dATP) results in significant enhancement of myofilament contraction. His group demonstrated that adenoviral mediated overexpression of ribonucleotide reductase (R1R2) increases [dATP] in adult rat cardiomyocytes, resulting in enhanced contraction and faster relaxation without altering Ca2+ release from the sarcoplasmic reticulum (SR). Thus, importantly, contraction efficiency of myofilaments is enhanced without altering cardiac Ca2+ cycling. A novel aspect of this therapy is that R1R2 overexpressing cardiac cells deliver dATP to non-transduced cardiomyocytes via gap junctions, increasing their contractility, thus indicating not all cardiomyocytes need to be transduced to achieve a therapeutic effect. To determine translational efficacy, Dr. Regnier and colleagues developed an adeno- associated viral vector with a cardiac specific promoter (cTnT455) to selectively overexpress both subunits of R1R2 in the heart using a single vector designated BB-R12 (AAV6-R1R2cTnT455). BEATBio is developing this technology to treat HF, and has made several major advancements demonstrating feasibility. In this project we will scale up the manufacturing of GLP-grade BB-R12, and to deliver BB-R12 to Yucatan mini-pig hearts 1 month following MI via coronary catheterization subsequent to the onset of HF. Cardiac function in vivo will be assessed by echocardiography and hemodynamic measurements at baseline, pre- administration, and at 1, 2, and 3 months post-administration. Hearts will then be removed for morphological and histological assessments, vector distribution and expression (multiple tissues), and cardiac tissue levels of dATP. We will also perform a long term study in infarcted mice to determine persistence of efficacy and safety. This study will provide information to guide more in depth preclinical studies on dose response, safety and efficacy, and aid in protocol development for Phase I human trials.